May 14, 2024
Version 3
Differentiation of Mesenchymal Stromal Cells to Endothelial-like cells in Spheroidal Culture V.3
- Simeng Li1,
- Isabel Arias Quiros1,
- Guenther Eissner1
- 1University College Dublin
Protocol Citation: Simeng Li, Isabel Arias Quiros, Guenther Eissner 2024. Differentiation of Mesenchymal Stromal Cells to Endothelial-like cells in Spheroidal Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82m17l2w/v3Version created by Simeng Li
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 15, 2024
Last Modified: May 14, 2024
Protocol Integer ID: 99749
Keywords: Mesenchymal stromal cells, Spheroids culture, endothelial differentiation, Spheroids disassociation
Funders Acknowledgements:
National Children's Research Centre (NCRC), Ireland
Grant ID: A-18-4
Abstract
In this protocol, an easy and cost-friendly method to form mesenchymal stromal cell (MSC) spheroids was specified. The MSC spheroids would form in 1-3 days and are suitable for endothelial differentiation. Accutase treatment can be used to disassociate the spheroids into single cells, for further analysis.
Multiple growth factors were used to differentiate MSC Spheroids into endothelial-like spheroids, which express endothelial markers such as von willebrand factors (vWF) and CD31 detected by immunofluorescence.
Materials
| A | B | C | |
| Reagent Name | Company/ Brand | Catalogue number | |
| 0.05% Trypsin-EDTA (1x) | Gibco | 25300-054 | |
| Trypsin Neutralizing Solution | Lonza | CC-5002 | |
| Recombinant human epidermal growth factor/EGF | PELOBiotech | C029-B | |
| Recombinant human fibroblast growth factor (bFGF) | PELOBiotech | C046-A | |
| Recombinant human vegf-a/vegf165 | PELOBiotech | C083-A | |
| Propidium Iodide Solution | Sigma-Aldrich | P4864 | |
| Mesencult-ACF plus culture kit | Stemcell Technologies | 5448 | |
| L-Glutamine (100x) | Gibco | 25030-024 | |
| IBIDI Angiogenesis Slides | ibidi | 81506 | |
| Cell culture plate, 96 wells, round bottom, untreated, individually wrapped | VWR | 392-0291 | |
| Dulbecco PBS, w/o Ca++/ Mg++ | Promocell | C-40232 | |
| Human serum from human male ab plasma | Sigma-Aldrich | H4522 | |
| ibidi mounting medium | ibidi | 50001 | |
| Trypan Blue Solution, 0.4% | Gibco | 15250061 | |
| PBS, pH 7.2 | Gibco | 20012027 | |
| TWEEN 20 | Sigma-Aldrich | P1379 | |
| Tris Buffered Saline (TBS), 10x | Sigma-Aldrich | T5912 | |
| Paraformaldehyde (PFA), 16% w/v, methanol free | Thermo Scientific Chemicals | 043368-9M | |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3294 | |
| Fetal Bovine Serum (FBS) | Sigma-Aldrich | F7524 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| Olympus FV1000 confocal microscope | Olympus | / | |
| Accuri C6 flow cytometer | BD Biosciences | / |
Mesenchymal stromal cells (MSC) culture
Mesenchymal stromal cells (MSC) culture
Coat culture flasks with Animal Component-Free Cell Attachment Substrate prior to cell seeding. Dilute Animal Component-Free Cell Attachment Substrate 1:15 in D-PBS (without Ca++ and Mg++) and coat the culturewares 2 hours at room temperature (15-25 °C), using the sufficient volume to cover the culture surface. Alternatively, coat flasks overnight at 2-8 °C with flask lid sealed by parafilm.
Bring flasks with attachment substrate to room temperature if flasks were coated at 2-8 °C. Remove attachment substrate completely by tilting the flask and gathering the substrate to the edge of the flask.
Wash flasks once with D-PBS. Then the flasks are ready to use.
Complete 500 mL MesenCult-ACF Plus Medium by adding 1 mL of MesenCult-ACF Plus 500X Supplement and 2 mM of L-glutamine.
Trypsinze MSCs with controlled treatment time. Normally 30s-1min is sufficient to detach MSC from flasks. Use Trypsin neutralizing solution to neutralize trypsin since there is no serum in the cell culture medium.
Count cells with Trypan Blue exclusion and seed MSC in fully-supplemented MesenCult-ACF Plus Medium.
Mesenchymal stromal cell spheroids formation
Mesenchymal stromal cell spheroids formation
Split and resuspend MSC in fully-supplemented MesenCult-ACF plus medium.
5m
Count cells with Trypan blue and dilute cell to 3x10e5/ml or 12x10e5/ml. Cells need to be over 95% viable in order to form spheroids.
10m
Seed 50uL of cell suspension to each well of a 96-well round-bottom plate. There will be 15,000 cells per well.
5m
Keep MSC in incubator for 1-3 days until one spheroid is formed in each well. Depending on the size of the spheroids, they can be observed either with eyes or underneath a microscope. Culture condition is 37 degree, 5% CO2 and 80% humidity.
MSC spheroids formation after 3 days in culture20 steps
Primary umbilical cord MSC and hTERT BMMSC (an immortalized bone marrow MSC cell line) were used in the experiment. MSC were seeded at 60,000 or 15,000 per well.
Visible spheroids formed in each well after 3 days of culture. However, more than one spheroids were formed in some wells, especially in the wells with 60,000 MSC seeded. This observation suggested 15,000 cells per well is a good starting concentration to form MSC spheroids.
Step 10 case.pdf
160KB
Endothelial differentiation of MSC spheroids
Endothelial differentiation of MSC spheroids
Make endothelial differentiation medium by adding 10ng/ml (final concentration) VEGF, EGF, bFGF and 2% (v/v) human serum (HS) into fully supplemented MesenCult ACF-plus medium.
5m
Carefully remove the media from each well, avoid the spheroid stucking at the end of the tips.
10m
Add 50ul of differentiation medium into each well. Keep some spheroids as untreated control by adding normal culture medium.
5m
Differentiation duration can be between 5-11 days. It can be determined by how advanced the differentiated cells need to be.
Immunofluorescence to check endothelial markers of differentiated spheroids
Immunofluorescence to check endothelial markers of differentiated spheroids
1h
1h
Remove medium in each well carefully. Before fixation and permeabilization, wash spheroids three times with PBS, 5 min each.
The same 96-well round-bottom plate was used for fixation. Fixation was done with 4% PFA for 1 hour at room temperature (RT). Fixation time varies depending on the size of spheroids.
1h
Wash spheroids with 100uL PBS per well three times, 5 min each.
15m
Block spheroids using blocking buffer (PBS with 3% FBS, 1% BSA, 0.5% Triton X-100 and 0.5% Tween) for 2 hours at RT on a shaker.
2h
Dilute primary antibodies 1:100–1:200 (or according to manufacturor's instruction) in the blocking buffer. Add 50ul of primary antibody to each well and incubated overnight at 2-8 °C. Primary antibodies produced from different species can be used together.
Remove solution from each well and wash Spheroids with PBST (0.05% Tween‱ 20 detergent in PBS) for 10 min at RT, three times.
30m
Add corresponding secondary antibodies at 1:250 in blocking buffer for 2 h at RT.
2h
Remove solution from each well and wash spheroids with TBST (0.1% Tween‱ 20 detergent in Tris-buffered saline), 20 min each time, 3 times.
1h
Stain Spheroids with 300nM DAPI for 20 min at 37 degree. Then wash the spheroids once with 50 uL PBST for 5 min.
25m
Use a 1 ml pipette to take the spheroid up together with the solution from each well. Transfer the spheroid to a well of ibidi µ-Slide 15 Well 3D.
10m
Remove PBST from each well of ibidi µ-Slide, add a drop of ibidi mounting medium to mount the spheroids. Control the amount of mounting medium added, so that the spheroids will be located at the bottom of each well.
20m
Image the spheroids with a Olympus FV1000 confocal microscope (Olympus Lifescience), or other suitable confocal microscope.
Immunofluorescence images of MSC spheroids4 steps
After 5 days of spheroids formation, hTERT-BMMSC spheroids were differentiated for 11 days before harvested for immunofluorescence analysis.
Endothelial differentiation of MSC spheroids expressed vWF and CD31, which the CD31 expression hadn’t been observed in 2-D MSC endothelial differentiation assay in the lab, even after 23 days of endothelial differentiation medium treatment.
Step 26 case.pdf
150KB
Disassociation of MSC from Spheroids for flow cytometry analysis
Disassociation of MSC from Spheroids for flow cytometry analysis
Harvest 10 spheroids from each condition into one 1.5 mL Eppendorf tubes. Centrifuge at 300g, 4 min. Then remove and discard the supernatant.
Add 400 uL accutase solution (Sigma/ Cat: 6964) to each tube and incubate the tube on a Thermomixer comfort (Eppendorf, Hamburg, Germany) at 37 °C, 1400 rpm for 10min. Use a 200uL pipette to pipette up and down for ten times to further disassociate the spheroids. This is considered as one disassociation cycle.
Repeat the disassociation cycle 1-3 times more until no visible cell clumps remaining in the tube.
Wash cells once with 1 mL PBS. Centrifuge at 300g for 5 min and then cells can be resuspend in flow cytometry buffer for antibody labelling.
Protocol references
1. Redondo-Castro, E., et al., Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates. Bio Protoc, 2018. 8(16).
2. Grasser, U., et al., Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis. Ann Anat, 2018. 216: p. 1-8.
3. Wimmer, R.A., et al., Human blood vessel organoids as a model of diabetic vasculopathy. Nature, 2019. 565(7740): p. 505-510.
4. Oswald, J., et al., Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells, 2004. 22(3):p. 377-84.
5. Gang, E.J., et al., In vitro endothelial potential of human UC blood-derived mesenchymal stem cells. Cytotherapy, 2006. 8(3): p. 215-27.
6. Konno, M., et al., Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture. Biochem Biophys Res Commun, 2010. 400(4): p. 461-5.